. Legionella pneumophila, the agent of Legionnaire's disease, causes a significant proportion of both community-acquired and nosocomial pneumonia in the U.S. each year. More than 90 percent of the 50,000-200,000 cases estimated to occur are caused by this species, although more than 30 Legionella species have now been identified. Legionnaire's disease is s syndrome characterized by typical signs and symptoms associated with bacterial pneumonia, but in addition, many patients experience extra-pulmonary manifestations of disease, including renal, kidney and central nervous system dysfunction. The histopathology of lung infection shows an fibrinopurulent process and tissue necrosis. Bacteria are seen in close association with macrophages. The mortality rate remains relatively high (15-20 percent) in spite of appropriate diagnosis and therapy. On e of the unique properties of L. pneumophila not found in other Legionella species which are intrinsically less virulent is a secreted metalloprotease. It is now known that the protease has hemolytic and cytotoxic properties and that a single gene encodes all these functions in one polypeptide. This gene has now been cloned and sequenced and bears a striking resemblance to Pseudomonas aeruginosa elastase. In nature Legionella sp. are found in water in association with amoeba and other protozoans in which they are able to grow. The ability of Legionella to replicate is affected by temperature and depends upon the degree of intrinsic "virulence" of the strain. In human infections and in experimental animals, L. pneumophila enters macrophages by a unique process. Virulent strains are able to prevent intracellular killing and grow to the extent that they are released from the cell whereupon progeny bacteria may infect other cells. Although Legionella is not considered to be a true obligate intracellular parasite, growth can only occur intracellularly. Dr. Tompkins proposes to examine the genetic determinants of Legionella which permit adaptation to these diverse intracellular environments by isolating, cloning and characterizing those genes that may be expressed in response to environmental signals. She will use models of intracellular parasitism, in amoeba and eukaryotic cells, to study the behavior of Legionella mutants and the effect of environmental factors on intracellular growth. Among the putative determinants of pathogenicity she will focus upon include the metalloprotease, flagella, and phenotypic virulence. Isogeneic mutants will be selected by gene exchange and transposon-mediated mutagenesis. They will study the expression and regulation of genes encoding these traits, and the effect of environmental factors, in vivo in amoeba and eukaryotic cells, comparing isogeneic mutants with wild-type strains. The other major aspect of this proposal is to examine the contribution of these genes to infection and disease in an animal model in order to compare and contrast the effect of these gene products in animal infection and during growth in protozoans and eukaryotic cells.